This invention is directed to cloning, sequencing and expressing heparinase II and heparinase III from Flavobacterium heparinum.
The heparin and heparan sulfate family of molecules is comprised of glycosaminoglycans of repeating glucosamine and hexuronic acid residues, either iduronic or glucuronic, in which the 2, 3 or 6 position of glucosamine or the 2 position of the hexuronic acid may be sulfated. Variations in the extent and location of sulfation as well as conformation of the alternating hexuronic acid residue leads to a high degree of heterogeneity of the molecules within this family. Conventionally, heparin refers to molecules which possess a high sulfate content, 2.6 sulfates per disaccharide, and a higher amount of iduronic acid. Conversely, heparan sulfate contains lower amounts of sulfate, 0.7 to 1.3 sulfates per disaccharide, and less iduronic acid. However, variants of intermediate composition exist and heparins from all biological sources have not yet been characterized.
Specific sulfation/glycosylation patterns of heparin have been associated with biological function, such as the antithrombin binding site described by Choay et al., Thrombosis Res. 18: 573-578 (1980), and the fibroblast growth factor binding site described by Turnbull et al., J. Biol. Chem. 267: 10337-10341 (1992). It is apparent from these examples that heparin's interaction with certain molecules results from the conformation imparted by specific sequences and not solely due to electrostatic interactions imparted by its high sulfate composition. Heparin interacts with a variety of mammalian molecules, thereby modulating several biological events such as hemostasis, cell proliferation, migration and adhesion as summarized by Kjellen and Lindahl, Ann Rev Biochem 60: 443-475 (1991) and Burgess and Macaig, Ann. Rev. Biochem. 58: 575-606 (1989). Heparin, extracted from bovine lungs and porcine intestines, has been used as an anticoagulant since its antithrombotic properties were discovered by McLean, Am. J. Physiol. 41: 250-257 (1916). Heparin and chemically modified heparins are continually under review for medical applications in the areas of wound healing and treating vascular disease.
Heparin degrading enzymes, referred to as heparinases or heparin lyases, have been identified in several microorganisms including: Flavobacterium heparinum, Bacteriodes sp. and Aspergillus nidulans as summarized by Linhardt et al., Appl. Biochem. Biotechnol. 12: 135-177 (1986). Heparan sulfate degrading enzymes, referred to as heparitinases or heparan sulfate lyases, have been detected in platelets (Oldberg et al., Biochemistry 19: 5755-5762 (1980)), tumor (Nakajima et al., J. Biol. Chem. 259: 2283-2290 (1984)) and endothelial cells (Gaal et al., Biochem. Biophys. Res. Comm. 161: 604-614 (1989)). Mammalian heparanases catalyze the hydrolysis of the carbohydrate backbone of heparan sulfate at the hexuronic acid (1.fwdarw.4) glucosamine linkage (Nakajima et al., J. Cell. Biochem. 36: 157-167 (1988)) and are inhibited by the highly sulfated heparin. However, accurate biochemical characterizations of these enzymes has thus far been prevented by the lack of a method to obtain homogeneous preparations of the molecules.
Flavobacterium heparinum produces heparin and heparan sulfate degrading enzymes termed heparinase I (E.C. 4.2.2.7) as described by Yang et al., J. Biol. Chem. 260(3): 1849-1857 (1985), heparinase II as described by Zimmermann and Cooney, U.S. Pat. No. 5,169,772, and heparinase III (E.C. 4.2.2.8) as described by Lohse and Linhardt, J. Biol. Chem. 267: 24347-24355 (1992). These enzymes catalyze an eliminative cleavage of the (.alpha.1.fwdarw.4) carbohydrate bond between glucosamine and hexuronic acid residues in the heparin/heparan sulfate backbone. The three enzyme variants differ in their action on specific carbohydrate residues. Heparinase I cleaves at .alpha.-D-GlcNp2S6S(1.fwdarw.4 4).alpha.-L-IdoAp2S, heparinase III at .alpha.-D-GlcNp2Ac(or2S)60H(1.fwdarw.4 4).beta.-D-GlcAp and heparinase II at either linkage as described by Desai et al., Arch. Biochem. Biophys. 306(2): 461-468 (1993). Secondary cleavage sites for each enzyme also have been described by Desai et al.
Heparinase I has been used clinically to neutralize the anticoagulant properties of heparin as summarized by Waugh and Zimmermann, Perfusion Rev. 1(2): 8-13, 1993. Heparinase I and III have been shown to modulate cell-growth factor interactions as demonstrated by Bashkin et al., J. Cell Physiol. 151:126-137 (1992) and cell-lipoprotein interactions as demonstrated by Chappell et al., J. Biol. Chem. 268(19):14168-14175 (1993). The availability of heparin degrading enzymes of sufficient purity and quantity could lead to the development of important diagnostic and therapeutic formulations.